Download Dopamine transporter: involvement in selective dopaminergic

DOI 10.1007/s00702-004-0203-2
J Neural Transm (2004) 111: 1267–1286
Dopamine transporter: involvement in selective
dopaminergic neurotoxicity and degeneration
A. Storch1 , A. C. Ludolph1 , and J. Schwarz2
1
2
Department of Neurology, University of Ulm, and
Department of Neurology, University of Leipzig, Germany
Received March 8, 2004; accepted July 16, 2004
Summary. The carrier molecule that transports dopamine (DA) into dopamine neurons by an electrogenic, Naþ - and Cl -transport-coupled mechanism is known as the dopamine transporter (DAT). This uptake system is
exclusively expressed in DA neurons with significantly higher levels of
DAT expression in cells of the substantia nigra pars compacta than those
of the ventral tegmental area and arcuate hypothalamic neurons. The expression density of DAT strongly correlates with the extent of DA cell loss in
Parkinson’s disease (PD). There are also DAT gene polymorphisms associated with PD. These data suggest a role of the DAT in the pathogenesis
of PD. Though selective for its respective neurotransmitter, the DAT can also
transport synthetic=natural analogues of the transmitter. Should such compounds interact with vital intracellular structures, their penetration into the
neuron might have significant consequences. This sequence of toxic events
could indeed demonstrated for the synthetic toxin 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP), which produces selective degeneration of DA
neurons characteristic of PD. Dopaminergic toxicity of its active metabolite
1-methyl-4-pyridinium (MPPþ ) is mediated by the DAT through accumulation into DA neurons, where it inhibits mitochondrial complex I activity.
Various endogenous and exogenous heterocyclic molecules, which are structurally related to MPTP=MPPþ, such as isoquinolines and b-carbolines, have
been reported to exhibit similar toxic properties on DA cells, which are
conferred by their uptake by the DAT. Taken together, there is large body
of evidence from morphological, molecular biological and toxicological studies indicating that the DAT might be responsible for the selectivity of DA
cell death in PD.
Keywords: Dopamine transporter, dopaminergic degeneration, neurotoxicity,
Parkinson’s disease.
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Abbreviations
C b-carboline, DA dopamine, DAT dopamine transporter, HPPþ haloperidol
pyridinium ion, HEK-293 human embryonic kidney 293 cells, HEK-hDAT
dopamine transporter transfected HEK-293 cells, 6-OHDA 6-hydroxydopamine,
IQ isoquinoline, MPPþ 1-Methyl-4-phenylpyridinium ion, MPTP 1-Methyl-4phenyl-2,3,6-tetrahydropyridine, PD Parkinson’s disease, SNpc substantia nigra
pars compacta, TaClo 1-trichloromethyl-1,2,3,4-tetrahydro-b-carboline, THC
1,2,3,4-tetrahydro-b-carboline, TIQ 1,2,3,4-tetrahydroisoquinoline.
Introduction
The dopamine transporter (DAT) belongs to a large family of Naþ - and Cl dependent transporters including other monoamine transporters, such as the
norepinephrine and serotonin transporter, as well as GABA and glycine carriers
(Amara and Kuhar, 1993; Giros and Caron, 1993). The transport process of the
DAT involves translocation of the substrate dopamine (DA) as well as two
sodium and one chloride ions across the cell membrane (Krueger, 1990;
McElvain and Schenk, 1992). The uptake is energetically coupled to transmembrane concentration gradient of Naþ , which is maintained by Naþ -Kþ -ATPases
(Hitri et al., 1994). The cDNAs encoding DAT have been cloned from various
species including rat, mouse, and human tissue (Kilty et al., 1991; Usdin et al.,
1991; Giros et al., 1992). The coding sequence for the DAT covers about 2 kb
corresponding to 617 to 630 amino acid residues, resulting in molecular
weights of the protein ranging from 60 to 97 kDa depending on glycosylation.
Comparison of the primary sequences of monoamine transporters revealed
between 69 and 80% homology between DAT and other monoamine transporters. Hydropathy analysis of the amino acid sequence of the DAT together with
immunofluorescence and electron microscopy investigations suggest the presence of 12 transmembrane domains (TM) with the C- and N-terminus within
the cytoplasm and a large extracellular loop between TM 3 and 4 (Fig. 1;
(Amara and Kuhar, 1993; Giros and Caron, 1993; Nirenberg et al., 1996).
In mammalian brain, DAT mRNA is localized in cell bodies and is restricted
to DA neurons (Amara and Kuhar, 1993; Augood et al., 1993; Lorang et al.,
1994; Nirenberg et al., 1996). DAT mRNA has been detected in the substantia
nigra (SN), the midbrain region and the brain stem with the highest expression
levels in the substantia nigra pars compacta and pars lateralis and ventral tegmental area (VTA) (Usdin et al., 1991; Shimada et al., 1992; Cerruti et al.,
1993b; Lorang et al., 1994). Co-localization with tyrosine hydroxylase immunostaining showed that the expression was localized only in cell bodies of DA
neurons (Usdin et al., 1991; Augood et al., 1993; Hurd et al., 1994; Lorang
et al., 1994). The first regional map of the distribution of the DAT protein in
postmortem human brain using [3H]mazindol showed DAT labeling most evident in the striatum and moderate labeling over DA cell bodies fields, including
the SNpc, VTA and the retrorubral field (Donnan et al., 1991). Generation of
antibodies highly specific to DAT allowed quantification and localization of the
DAT protein (Nirenberg et al., 1996; Hersch et al., 1997; Ciliax et al., 1999).
DAT immunoreactivity was concentrated in somatodendritic and terminal fields
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Fig. 1. Putative topology and structural features of the DAT protein. As suggested by the
primary sequence of the cDNA, the protein has 12 transmembrane domains (TM) with both
N- and C-termini located with the cytoplasm. Amino acids residues reported to be important for
the affinities of dopamine, 1-methyl-4-phenylpyridinium (MPPþ ) and cocaine derivative ()-2bcarbomethoxy-3b-(4-fluorophenyl)-tropane (CFT; inset) at the DAT molecule are illustrated with
colored boxes (labeling according to the sequence of the human DAT published by (Giros et al.,
1992). The amino acid residues were identified using site-directed mutagenesis and subsequent
binding=uptake studies (see text for details)
of mesencephalic DA neurons with most pronounced immunoreactivity in striatum and nucleus accumbens. The VTA and other brain areas with established
DA circuitry, such as the hypothalamic neurons, were stained less intensively.
In human brain samples, similar levels of DAT expression were detected in
somatodendritic and axonal domains of most mesotelencephalic DA neurons
(Miller et al., 1997; Ciliax et al., 1999).
The major physiological role of DAT is the termination of neurotransmission by rapid reuptake of DA from the synaptic cleft into presynaptic terminals,
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and it is believed to control the intensity and duration of DAergic neurotransmission by setting the concentration of DA in the extracellular space. Depending on cellular conditions, DAT is able to transport DA bi-directionally, but at
normal membrane potential and Naþ gradient the inward transport is greater
than the outward transport (Hitri et al., 1994). The function of DAT is regulated
or influenced by several other cellular factors and proteins including the presynaptic protein a-synuclein (Kitayama et al., 1994; Huff et al., 1997; Lee et al.,
2001a). Pharmacological characterization of cloned monoamine transporters
expressed heterologously have confirmed substrate specificity for the respective
neurotransmitter as shown earlier in synaptosomal preparations (for review, see
Amara and Kuhar, 1993; Giros and Caron, 1993; Hitri et al., 1994). Subsequently, several highly specific DAT inhibitors could be established such as
diphenyl-piperazine derivatives (for example GBR12935 or GBR12909). The
binding site of DA at the transporter molecule was initially investigated using
chimeric dopamine-norepinephrine transporters (Buck and Amara, 1994). It
includes an amino terminal domain (TM1–TM3) and with lower significance
TM10–TM11. These data were confirmed using site-directed mutagenesis and
subsequent binding=uptake studies using the mutated transporters in mammalian expression systems (Kitayama et al., 1992b, 1993, 1996c, 1998; Mitsuhata
et al., 1998; Lin et al., 1999, 2000a, b; Lee et al., 2000; Lin and Uhl, 2002).
Mutations of several amino acid residues in TM1–7 could be identified to
reduce binding affinity of DA at the DAT molecule (Fig. 1). On the other hand,
the binding sites of the cocaine derivative ()-2b-carbomethoxy-3b-(4-fluorophenyl)-tropane (CFT) were extensively characterized (Fig. 1; Kitayama et al.,
1992b, 1996c; Lin et al., 1999, 2000a, b; Lee et al., 2000; Lin and Uhl, 2002,
2003). The displayed amino acid residues significantly influence the affinities of
the respective substance, but from the site-directed mutagenesis experiments it
is not clear whether all of these residues are located within the binding sites of
the compounds or whether they affect the binding in an allosteric manner.
However, some of the positions of amino acid residues lie close together while
other amino acid residues are fare away from each other in the primary
sequence. On the other hand, these residues could lie next to each other in
the mature DAT structure even if they are not nearby in primary sequence.
Interestingly, there are some mutations selectively alter the affinity of cocaine
analogs, but spare their affinities for DA (compare Fig. 1 with inset; for review
see Uhl and Lin, 2003).
In this review we discuss the importance of the DAT molecule for the
selective vulnerability of dopaminergic cells against specific neurotoxins as
well as in Parkinson’s disease (PD). Thus, we summarize the data on the importance of the DAT molecule for the selectivity of neurotoxins such as 6-hydroxydopamine, 1-methyl-4-phenylpyridinium (MPPþ ) and structurally related
compounds towards DAT expressing cells. We include a discussion on structural requirements of such compounds for both cellular uptake by the DAT and
DAT-mediated cytotoxicity. Since the DAT molecule is discussed to be involved
in the selectivity of dopaminergic degeneration in PD, we compare these toxicological data with evidences for the importance of the DAT in the etiopathogenesis of PD.
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Importance of the DAT for selective dopaminergic toxicity
Although DAT is selective for its respective neurotransmitter DA, it can also
transport synthetic or natural structural analogues of the neurotransmitter.
Should such compounds interact with vital intracellular functions or structures,
their penetration into the cell via the DAT might have significant consequences.
The importance of the DAT for the selectivity of toxicity of such structural
analogues of DA will be discussed in detail in the following sections (please
refer to Fig. 2 for chemical structures).
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
and 1-methyl-4-phenylpyridinium (MPPþ )
The potent exogenous neurotoxin MPTP produces a Parkinson-like syndrome
in mammalians by causing a selective degeneration of DA neurons in the SNpc
with eosinophilic inclusions (Langston et al., 1983). After entering the CNS,
MPTP is metabolized largely in astrocytes by monoamine oxidase type B into
1-methyl-4-phenyl-1,2,3-dihydropyridinium (MPDPþ ), which spontaneously
undergoes oxidation to MPPþ (Chiba et al., 1984; Przedborski et al., 2000).
Russ and co-workers provided evidences that MPPþ uses the extraneuronal
monoamine transporter (EMT) to depart glial cells (Russ et al., 1996). Once
inside the dopaminergic cell, MPPþ induces a deleterious cascade of events
include mitochondrial respiration deficit, oxidative stress, and energy failure
(Di Monte et al., 1986; Ramsay and Singer, 1986; Kutty et al., 1991; Storch
et al., 1999, 2000a; Przedborski et al., 2000). In recent studies it becomes
evident that other proteins, such as wild-type and PD related mutant a-synucleins, modulate MPTP=MPPþ toxicity towards DA neurons both in vivo and
in vitro (Lee et al., 2001a; Dauer et al., 2002; Lehmensiek et al., 2002; Sidhu
et al., 2004).
Fig. 2. Structures of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4phenylpyridinium (MPPþ ), dopamine (extended form), 6-hydroxydopamine as well as isoquinoline and b-carboline derivatives
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The selectivity of MPTP for DA neurons is considered to be a consequence
of active uptake of its metabolite MPPþ into DA neurons via the DAT (Chiba
et al., 1985; Javitch et al., 1985). MPPþ has high affinity for the DAT with Km
values of about 0.05 to 0.1 mM in rat synaptosomes, but 20 mM in cell lines with
ectopic expression of DAT (Javitch et al., 1985; Pifl et al., 1993; Storch et al.,
2004). It is taken up rapidly with Vmax values comparable to those measured for
DA. Cellular uptake of MPPþ via the DAT shows a similar pharmacological
profile compared to that of DA uptake. Consistently, the expression of DAT
confers toxicity of low concentrations of MPTP=MPPþ towards neuronal cells
in vivo and in vitro. Mice lacking the DAT gene do not show MPTP-induced
DAergic toxicity in vivo (Gainetdinov et al., 1997; Bezard et al., 1999), whereas
mice with increased levels of DAT expression are more sensitive to MPTP
(Donovan et al., 1999). Selective blockers of the DAT, such as mazindol, are
reported to completely prevent MPTP-induced nigrostriatal toxicity, while inhibitors of the noradrenaline transporter fail to protect against MPTP toxicity
(Javitch et al., 1985; Ricaurte et al., 1985; Mayer et al., 1986). Consistently,
MPTP=MPPþ show strong toxicity towards neuronal cell lines expressing the
DAT (Kutty et al., 1991; Bloomquist et al., 1994; Storch et al., 2000a). These
results were confirmed by toxicological studies on several neuronal and nonneuronal heterologous expression systems of the DAT (Kitayama et al., 1992a,
1998; Pifl et al., 1993, 1996; Storch et al., 1999). Using mutated DAT expressed
in heterologous expression systems, Kitayama and co-workers demonstrated
that sensitivity to MPPþ toxicity correlates closely with the affinity (Km value)
of MPPþ at the transporter, but does not correspond to uptake velocity
(Kitayama et al., 1998). On the other hand, several studies report high uptake
velocity or turn over number (Vmax=maximal binding sites) of MPPþ by the
DAT, but low turn over number by the noradrenaline transporter compared to
their natural substrates. In contrast, apparent affinity for MPPþ at the noradrenaline transporter is higher than that at the DAT protein (Buck and Amara,
1994; Pifl et al., 1996). These latter data suggest that high Vmax of MPPþ by
the DAT might contribute to the selectivity of DAergic toxicity of MPPþ.
Kitayama and co-workers demonstrated bi-directional transmembrane
transport of MPPþ through the DAT (Kitayama et al., 1993, 1996a). Using
mutant DAT with different transport properties ectopically expressed in COS
cells, Kitayama and co-workers (1998) showed that indeed the ratio of influx=
efflux turnover through the transporter may be an important factor for MPPþ
toxicity (Kitayama et al., 1998). Some mutations of the DAT protein resulting
in enhanced reverse transport compared to that of wild type DAT (Y273A;
double mutant S353A=S335A; Y533F) leads to decreased sensitivity of the
cells against MPPþ toxicity (Kitayama et al., 1998). The binding site of MPPþ
at the transporter molecule was initially investigated using chimeric dopaminenorepinephrine transporters (Buck and Amara, 1994). In contrast to DA, not
only the amino terminal domain spanning TM1–TM3, but also a domain
including TM10–TM11 significantly influences apparent affinity of MPPþ
at the DAT. The above mentioned site-directed mutagenesis approach confirmed these data (see Fig. 1 for details; Kitayama et al., 1993, 1998; Mitsuhata
et al., 1998; Lin et al., 1999; Lee et al., 2000). Dopamine and MPPþ probably
Dopamine transporter and selective dopaminergic vulnerability
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recognize overlapping, but different binding sites at the DAT molecule. Taken
together, these data clearly demonstrate that the DAT is responsible for the high
sensitivity and selectivity of MPTP=MPPþ toxicity towards DA neurons both
in vitro and in vivo. However, it remains unclear whether affinity of MPPþ at the
transporter or turn over number is more important for cellular sensitivity to
MPTP=MPPþ toxicity. Recent studies demonstrated that mutant a-synucleins
related to PD further increase DAT-mediated selectivity of MPPþ toxicity, most
likely by direct functional coupling to the DAT molecule (Lee et al., 2001b;
Lehmensiek et al., 2002).
Pyridine derivatives structurally related to MPTP=MPPþ
Michel and co-workers (1989) studied the toxic effects of 13 potential environmental pyridinium compounds structurally related to MPTP=MPPþ on mesencephalic DA neurons from rat in vitro (Michel et al., 1989). Among the group of
these pyridinium compounds, only two very closely related compounds (2methyl-MPPþ and p-amino-MPPþ ) showed selective effects against DA neurons, but were less effective and less selective compared to MPPþ. There are no
uptake studies available with these substances. Haloperidol, a widely used antipsychotic compound with a pyridine structure is metabolized to haloperidol
pyridinium ion (HPPþ ) by cytochrome P450, a reaction similar to the conversion of MPTP to MPPþ. It has been proposed that long-term extrapyramidal
symptoms, such as tardive dyskinesia, could be caused by neurotoxic effects of
HPPþ or other metabolites of haloperidol following uptake by monoamine
transporters. Indeed, HPPþ and other metabolites are shown to be toxic in vitro
to rat mesencephalic cultures and neuroblastoma cells (Bloomquist et al.,
1994). Uptake studies on DAT, NET and SERT using transfected COS-7 cells
and mouse synaptosomal preparations revealed that haloperidol, HPPþ and
their tetrahydropyridinium analogs inhibit all monoamine transporters without
relevant differences of potencies (Bryan-Lluka et al., 1999). Accordingly, using
heterologous expression systems we were not able to show DAT-dependent
toxicity of HPPþ (unpublished results). These data suggest that haloperidol
and its analogs are not likely to cause neurotoxicity by a mechanism similar
to that of MPTP=MPPþ involving the uptake by monoamine transporters.
Isoquinoline derivatives
Isoquinoline derivatives refer to isoquinoline (IQ) itself, various reduced species, like 3,4-dihydro-IQs and 1,2,3,4-tetrahydroisoquinoline (TIQ), and their
substituted congeners (e.g. 1-benzyl-TIQ). They are widely distributed in the
environment and occur naturally in mammalian brain where they are synthesized by an enzymatic and non-enzymatic Pictet-Spengler condensation of biogenic amines with aldehydes (Melchior and Collins, 1982). IQs are metabolized
by cytochrome P450, N-methyltransferases and monoamine oxidases leading to
charged quaternary forms (isoquinolinium cations; McNaught et al., 1998).
Interestingly, there are evidences for altered levels of DA-derived catecholic
IQs, in particular derivatives of 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol) and the synthesizing enzymes in patients with PD
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(Maruyama et al., 1996; Naoi et al., 1998). Since many IQ derivatives are
structurally related to MPTP=MPPþ (refer to Fig. 2), IQs are considered as
endogenous and=or exogenous DAergic neurotoxins playing a role in the etiopathogenesis of PD (McNaught et al., 1998).
The partial selectivity of IQ derivatives towards DAergic cells is considered
to be a consequence of cellular uptake by the DAT as shown for MPPþ. Indeed,
several IQ derivatives show partially selective but weak cytotoxicity in DAergic
cells lines and primary DA neurons (McNaught et al., 1996a; Goto et al., 1997;
Maruyama et al., 1997; Takahashi et al., 1997; Storch et al., 2000a). In vivo
studies using experimental animals including non-human primates demonstrated that some IQs are able to produce a parkinsonian syndrome with neurochemical, histological and behavioural changes after chronic treatment (for
review, see McNaught et al., 1998). To confirm the importance of the DAT as
well as the structural requirements for DAT-mediated toxicity induced by IQ
derivatives we used non-neuronal and neuronal heterologous expression systems of DAT, which are extremely sensitive to very low concentrations of
MPPþ (Storch et al., 1999, 2002). Out of 21 IQ derivatives, only the 2[N]methylated compounds showed enhanced cytotoxicity in both DAT expressing
cell lines with 2 to 14-fold reduction of half-maximal toxic concentration
(TC50 values) compared to parental cell lines. The rank order of selectivity in
both cell systems was: MPPþ 2[N]-methyl-IQþ > 2[N]-methyl-norsalsolinol
(2[N]-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) ¼ 2[N]-methylsalsolinol. The selective toxicity of these compounds can be blocked by DAT
inhibitors, such as GBR12909.
Neutral and quaternary IQs are able to partially inhibit [3H]DA accumulation into striatal synaptosomes from rat, but they are far less potent than MPPþ
(IC50 values in the micro- to millimolar range versus less than 1 mM; (Heikkila
et al., 1971; Hirata et al., 1990; McNaught et al., 1996b). The most potent
blockers of [3H]DA uptake are TIQ derivatives with catechol structures. Using
tritiated substances, Heikkila and co-workers showed accumulation of 6,7-dihydroxy-1,2,3,4-TIQ and 1-methyl-4,6,7-trihydroxy-1,2,3,4-TIQ in rat striatal
synaptosomes (Heikkila et al., 1971). Takahashi and co-workers studies the
uptake characteristics of catecholic IQs in SH-SY5Y cells using a HPLC-based
method (Takahashi et al., 1994). They found that only the (R)-isomer of 2[N]methyl-salsolinol is transported by the DAT, while its (S)-isomer, salsolinol and
1,2-dimethyl-6,7-dihydroxyisoquinolinium are not. DA and other DAT inhibitors block the uptake of 2[N]-methyl-(R)-salsolinol. Similarly, 2[N]-methyl-IQþ
has been shown to accumulate in rat striatal synaptosomes (Hirata et al., 1990).
Another extensive study using rat striatal synaptosomes revealed that 2[N]methyl-IQþ , (R)-salsolinol and 2[N]-methyl-(R)-salsolinol are accumulated via
the DAT (Matsubara et al., 1998b). Quaternization of TIQ’s reduced their
original properties as substrates for the DAT. Overall the data discussed here
demonstrate that several IQs, in particular 2[N]-methylated catecholic IQs, are
substrates for the DAT with both moderate to low affinities at the DAT and
moderate uptake velocities compared to MPPþ and DA.
We suggest that 2[N]-methylated IQ derivatives structurally related to
MPTP=MPPþ are selectively toxic to DA neurons via uptake by the DAT.
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However, low affinity and uptake velocity of IQs at the DAT may be a ratelimiting factor for DAergic toxicity. Future studies are warranted to compare
uptake kinetics and toxicity of IQs mediated by other monoamine transporters
to further confirm the role of DAT-mediated uptake for their DAergic toxicity
(Buck and Amara, 1994; Pifl et al., 1996).
-Carboline derivatives
b-Carbolines (bCs; pyrido-indoles) are heterocyclic molecules occurring both
exogenously and endogenously (for review, see Collins and Neafsey, 2000),
which are structurally related to the parkinsonian neurotoxin MPTP=MPPþ
(for structures refer to Fig. 2). Some of these bCs are reported to cause nigrostriatal neurodegeneration and behavioral impairment in several animals, including non-human primates (Collins et al., 1986; Matsubara et al., 1998a), most
likely by sequential biotransformation via N-methylations to b-carbolinium
ions (Waring et al., 1989; Collins et al., 1996; Collins and Neafsey, 2000).
These data are consistent with in vitro studies showing cytotoxicity of N-methylated b-carbolinium ions in primary mesencephalic cultures from rat with partial
selectivity for DA neurons (Collins et al., 1996; Matsubara et al., 1998a). In
HEK-293 cells expressing the DAT, only 2[N]-methylated compounds showed
enhanced cytotoxicity with 1.3 to 4.5-fold reduction of TC50 values compared
to the parental cell line (Storch et al., 2004). The rank order of selectivity was:
MPPþ 2[N],9[N]-DiMe-harminium > 2[N]-Me-harminium > 2[N],9[N]-DiMeharmanium ¼ 2[N]-Me-norharmanium > 2[N]-Me-harmanium > 2[N],9[N]-DiMenorharminium. These results in HEK-hDAT cells exposed to 2[N]-methylated
and 2[N],9[N]-dimethylated bCs parallel the toxicity of the same compounds
towards DA neurons in fetal rat mesencephalic cultures (Cobuzzi et al., 1994;
Collins et al., 1995, 1996; Matsubara et al., 1998a).
As for MPPþ, cellular uptake of bCs or rather b-cabolinium ions by the DAT
is considered to be involved in the selectivity of DAergic toxicity of these
compounds. Indeed, Drucker and co-workers (1989) showed that 2[N]-methylated b-carbolinium ions are able to inhibit [3H]DA uptake into rat striatal
synaptosomes, but IC50 values are lower compared to MPPþ (12 to approx.
150 mM for b-carbolinium ions and 0.5 mM for MPPþ, respectively; Drucker
et al., 1990). Furthermore, the partially competitive nature of inhibition two
effective compounds, 2-methyl-harmine and harmine, indicate that uptake of
the b-carbolines is mediated in part by synaptosomal DAT (Drucker et al.,
1990). This view is further supported by the fact that accumulation of
2-[14C]methyl-harmane into synaptosomes is partially blocked by the potent
DAT inhibitor nomifensine (Drucker et al., 1990). Using the same experimental
setup, Matsubara and co-workers measured the uptake of the neutral b-carboline norharmane and quaternary b-carboliniums 2-methyl-norharmanium
and 2,9-dimethyl-norharmanium, respectively, using a HPLC-based method
(Matsubara et al., 1998b). They found that norharmane is an unfavorable substrate
for the DAT, while the N-methylated compounds showed DAT mediated influx
into striatal synaptosomes with 1–4 times higher Km values and 10 times lower
uptake velocity (Vmax). We established a novel uptake assay in HEK-293
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cells expressing the DAT utilizing the fluorescent properties of bCs (Storch
et al., 2004). Out of 12 non-methylated and methylated bC derivatives (refer
to Fig. 2 for structures) all 2[N]-methylated bCs were transported into the cell
through the DAT with up to 5 times higher Km and 12 to 220 times lower
Vmax values compared to DA and MPPþ (Storch et al., 2004). Compared to
the toxicity data in the same cell line, there is a weak correlation of DATmediated selectivity with the affinity of bCs at the DAT (Km values), but not
with Vmax. These data suggest that DAT-mediated cellular uptake of 2[N]methylated bCs is most likely responsible for their selective toxicity towards
DA neurons.
The halogenated b-carboline derivative 1-trichloromethyl-1,2,3,4-tetrahydro-b-carboline (TaClo) is formed under physiological conditions via the
Pictet-Spengler condensation from the biogenic amine tryptamine and the
synthetic aldehyde chloral (Bringmann et al., 1995). Indeed, several studies
provide evidences for monoaminergic degeneration in vivo (Bringmann et al.,
1995; Gerlach et al., 1998). TaClo is able to inhibit DA and serotonin
uptake with IC50 values in the mM range. Furthermore, in vitro toxicological
studies using DAergic and serotoninergic cell lines (IMR-32 and JAR,
respectively) revealed that TaClo penetrates cell membranes by passive
diffusion and not by selective uptake systems (Bringmann et al., 2000).
Consistently, there is no DAT-mediated toxicity and uptake of TaClo or its
N-methylated derivative in HEK-293 cells expressing DAT (unpublished
observations).
6-Hydroxydopamine
6-hydroxydopamine (6-OHDA) is a toxin selective for catecholaminergic cells
in vivo, which is widely used to produce animal models of PD (Breese and
Traylor, 1970; Jonsson and Sachs, 1971; Kostrzewa and Jacobowitz, 1974). 6OHDA can be formed from DA by non-enzymatic hydroxylation in presence of
Fe2þ and H2O2. Moreover, Andrew et al. (1993) recently detected endogenous
6-OHDA in urine samples of patients suffering from PD, suggesting that this
compound may be involved in the pathogenesis of PD (Andrew et al., 1993).
The partial selectivity of 6-OHDA toxicity in vivo towards catecholaminergic
neurons was attributed to its uptake via catecholamine transporter systems,
including the DAT (Jonsson and Sachs, 1971; Kostrzewa and Jacobowitz,
1974). Furthermore, Cerruti and co-workers (1993) showed partial protection
against 6-OHDA toxicity by selective and potent DAT inhibitors in cultured DA
neurons (Cerruti et al., 1993a). On the other hand, several studies using different blockers of monoamine transporters as well as heterologous expression
systems of the DAT suggest that 6-OHDA toxicity in vitro is not mediated by
transmembrane uptake via catecholaminergic uptake systems (Abad et al.,
1995; Storch et al., 2000b). Furthermore, in vitro studies using different catecholaminergic and non-catecholaminergic primary cell and cell lines revealed
no selectivity of 6-OHDA-induced toxicity for catecholaminergic cells (Michel
et al., 1990; Abad et al., 1995; Storch et al., 2000b). 6-OHDA shows only low
affinity at the DAT (Michel et al., 1990; Decker et al., 1993) and no detectable
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levels of 6-OHDA were found in cytosolic extracts of PC12 or SH-SY5Y cells
following incubation with up to 600 mM 6-OHDA (Decker et al., 1993). The
reasons for the discrepancies between the in vivo and in vitro experiments
remain unclear.
Structural requirements for DAT-mediated uptake and toxicity
The herein reviewed studies on DAT-mediated toxicity and cellular uptake
of azaheterocyclic molecules provide significant information on structureactivity=toxicity (selectivity) relationships. The intermolecular distance between the N-atom and the centrinoid of the benzene or catechol ring is one
of the most important factors for the affinity of compounds at the DAT
molecule. The conformation of the DA molecule at the uptake site of the
DAT is the extended or trans form (Horn, 1974; Meiergerd and Schenk,
1994). The distance between the N-atom and the centrinoid in MPPþ and
b-carboline derivatives is very close to that of the extended DA conformation.
In contrast, this distance is much shorter in IQ derivatives, which is most
likely responsible for their lower affinity and uptake velocity at the DAT and
subsequent lower selectivity of toxicity as compared to MPPþ and bC derivatives (Storch et al., 2002, 2004). The charge of the N-atom seems to be
another factor not only for the affinity of the compounds to the transporter
molecule, but also for the transmembrane transport of the compounds by the
DAT (Matsubara et al., 1998b). Indeed, DAT-mediated toxicity and uptake
were restricted to quaternary 2[N]-methylated compounds with a net charge
comparable to DA, whereas structurally identical neutral compounds lack
selective toxicity towards DAT-expressing cell lines (see Fig. 2). Thus,
2[N]-methyl-IQþ , which has the closest structure-based relationship to MPPþ
within the IQ derivatives (no side chains; equivalent charge of the N-atom of
the pyridine ring), showed the highest selectivity towards DAT-expressing
cells. All modifications of the original structure of 2[N]-methyl-IQþ resulted
in partial or complete loss of selective toxicity towards DAT-expressing cells.
Among the N-methylated IQ derivatives, catecholic fully oxidized IQs (TIQ
derivatives) showed less selectivity for DAT-expressing cells compared to
2[N]-Me-IQþ . In agreement with Kawai and co-workers (Kawai et al.,
1998), methoxylation of carbonyl residues 6 and 7 of the benzene ring, as
in 2[N]-Me-norsalsolidine, completely abolished DAT-mediated toxicity.
While methylation at position 1 of the pyridine ring does not alter toxicity=
selectivity for DAT-expressing cells, large side chains at this position seems
to inhibit DAT-dependent toxicity. In N-methylated bC derivatives, an addition of a methoxy group on position 7 of the aromatic ring increases the
DAT-mediated toxicity, but not their uptake kinetics at the DAT molecule.
Systematic comparison of DAT-mediated toxicity induced by bCs with their
uptake kinetics suggests that the affinity of azaheterocyclic compounds at the
DAT is the major factor for their toxic selectivity towards DAT-expressing
cells. Thus, the lower affinities (greater Km values) of bCs and IQs at the
DAT compared to that of MPPþ might explain the moderate selectivity of
these compounds towards DA neurons.
1278
A. Storch et al.
Importance of DAT in the etiopathogenesis of Parkinson’s disease
MPTP produces a Parkinson-like syndrome in human and non-human primates
with remarkable similarities to PD (Langston et al., 1983; Forno et al., 1986).
Langston and co-workers recently reported evidences of active neurodegeneration in the SNpc in humans years after MPTP exposure in humans, suggesting
that MPTP intoxication may lead to a progressive disorder of the nigrostriatal
pathway (Langston et al., 1999). As described above, there are many exogenous
and endogenous compounds structurally related to MPTP=MPPþ showing toxicity in the central DAergic system, leading in part to parkinsonian syndromes.
This large body of evidences for the pivotal role of the DAT in selective vulnerability of DA neurons against parkinsonism-inducing compounds leads to
extensive morphological and genetic investigations on the importance of the
DAT molecule in PD.
Morphological studies
Three groups of DA neurons in different regions show substantially different
extent of neuronal cell loss in PD brains. DA neurons of the arcuate nucleus of
the hypothalamus providing the tuberoinfundibular DAergic system display no
detectable loss in PD. DA neurons of the VTA those provide DAergic innervation
of the cerebral cortex and limbic forebrain show only 40–60% cell death in PD
brains (Uhl et al., 1985; German et al., 1989). On the other hand, DA neurons
within the SNpc providing the dense DAergic projections to the striatum display
the highest degree of degeneration in PD brains (Waters et al., 1988; Pakkenberg
et al., 1991). Consistently, DA uptake sites have been found to be markedly
decreased in the striatum in patients with PD both postmortem and in vivo
(Janowsky et al., 1987; Leenders et al., 1990; Miller et al., 1997; Tatsch et al.,
1997). These data document a strong correlation between the density of DAT
expression and the extent of DA cell losses in PD brains (SNpc>VTA>arcuate
nucleus in both cases). There are some intact DA neurons that lack Lewy body
pathology in the SNpc from PD patients showing average DAT mRNA levels
lower than those of age- and gender-matched controls (Uhl et al., 1994). DAT
studies in living humans using PET or SPECT combined with autoradiographic
studies of the DAT in tissue samples obtained post mortem suggest that there is a
significant individual variation in the DAT expression, which is not related to age
known to influence the DAergic system (Staley et al., 1995): Subjects of similar
age can display up to 2-fold differences in radioligand binding to the DAT. These
differences are in the same range as those that can significantly alter the susceptibility to DAergic toxins in many transgenic mice models of the DAT (Donovan
et al., 1999). However, these morphological data suggest that DAT-based microcompartmentalization of DA or other toxins in DA neurons may be involved in
selective neurodegeneration in PD.
Molecular biology and genetic studies
Studies of the large human gene for the DAT on the short arm of chromosome 5
(5p 15.3) reveal several polymorphisms (Giros et al., 1992; Sano et al., 1993;
Morino et al., 2000). Studies of these polymorphisms could identify allelic gene
Dopamine transporter and selective dopaminergic vulnerability
1279
variants that could contribute to genetically determined human individual differences in both protein coding sequences and levels of DAT expression. These
individual differences could provide plausible sources for differential vulnerabilities to DAergic toxicities. However, association studies of these polymorphisms with PD revealed conflicting results: Two independent research
groups demonstrated a significant association of a single nucleotide polymorphism in exon 9 (1215A=G) with PD (Morino et al., 2000; Nishimura et al., 2002),
but two other groups were not able to confirm these results (Kimura et al., 2001;
Lin et al., 2002). The DAT gene contains a 40 base pair variable number tandem
repeat (VNTR) in the 3’ untranslated region of the gene (Sano et al., 1993). Le
Couteur and co-workers (1997) reported an association of this DAT gene VNTR
with PD in Caucasians (Le Couteur et al., 1997), but not in a Chinese population (Leighton et al., 1997; Lin et al., 2002): The rare 11-copy allele of the DAT
VNTR (0.3% frequency of all alleles) increases the risk of PD about 10-fold in
Caucasians (Le Couteur et al., 1997). This result is of some interest, because the
DAT VNTR sequence includes possible binding regions for several transcription factors, and indeed VNTR polymorphism effects translation of the DAT
protein in vivo (Heinz et al., 2000). However, such an increase in the 11-allele
of the DAT gene was not observed in two other studies in caucasian populations
(Plante-Bordeneuve et al., 1997; Mercier et al., 1999). Interestingly, the homozygote 10-copy genotype of variable number tandem repeat dopamine transporter gene might confer protection against PD for male, but not to female
patients (Lin et al., 2003). The reasons for these differences are unclear, but
most likely occurred as a result of methodological problems such as selection
bias, false positive results because of small study populations and=or multiple
testing without adjustment. Furthermore, geographic variations in toxins and
toxin exposure, variations in metabolic genotypes between the populations
might be responsible for different results in the genetic studies. Together, the
association studies concerning the DAT gene and the risk of PD did not reveal
conclusive results.
Conclusions
The in vivo and in vitro data show that the DAT plays a pivotal role for the
selective DAergic toxicity of endogenous and exogenous N-methylated heterocyclic molecules structurally related to MPTP or its active metabolite MPPþ
and that the DAT may be important for selective degeneration of the DA neurons found in PD. Epidemiological studies indicate that a number of environmental factors increase the risk of developing PD. However, no specific toxin
has been found in the brain of PD patients, and in many instances the parkinsonism produced by toxins is not that of typical Lewy body PD. Nevertheless,
recent findings of altered levels of potential DAergic neurotoxins, such as IQ
derivatives, in patients with PD together with the finding that the level of
degeneration in PD closely corresponds with the level of cellular DAT expression further support the hypothesis that miscompartmentalization of toxic compounds by the DAT might be responsible for the selectivity of DAergic
degeneration found in PD. However, genetic association studies on the DAT
1280
A. Storch et al.
gene in PD did not reveal conclusive results and future studies have to clarify
the importance of interactions of the DAT gene or alterations of function=
expression of the DAT protein with environmental factors for the pathogenesis
of PD. The identification of a DAT-based mechanism that explains selective DA
cell loss in PD could have several implications: Compounds that inhibit intracellular uptake mediated by DAT would be of interest for therapeutic trials
targeted towards slowing the disease onset and=or progression. Such agents
could modulate transporter function, for example by altering the phosphorylation of DAT (Kitayama et al., 1994; Huff et al., 1997), or act directly on the
DAT molecule. As demonstrated above, DA and MPPþ probably recognize
overlapping, but different binding sites at the DAT molecule, suggesting a
possible strategy for developing drugs which specifically block uptake or stimulate release of potential DAergic neurotoxins via the DAT without affecting
DA uptake (as demonstrated already for cocaine derivatives and other DAT
blockers, showing differential effects on the release of MPPþ and DA, respectively, through the DAT (Kitayama et al., 1996b). Furthermore, individual differences of DAT expression, genetically or environmentally determined, could
serve as markers for susceptibility for PD.
Acknowledgements
Supported in part by the University of Ulm Research Foundation (to A.S.) and the Ministerium
f€
ur Wissenschaft, Forschung und Kunst Baden-W€urttemberg, Germany (to A.S. and J.S.).
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Authors’ address: A. Storch, M.D., Department of Neurology, Technical University of
Dresden, Fetscherstrasse 74, 01307 Dresden, Germany, e-mail: [email protected].
tu-dresden.de